Recombinant Treponema pallidum rare outer membrane protein 1 (Tromp1) expressed in Escherichia coli has porin activity and surface antigenic exposure

David R. Blanco, Cheryl I. Champion, Maurice M. Exner, Ellen S. Shang, Jonathan T. Skare, Robert Hancock, James N. Miller, Michael A. Lovett

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

We recently reported the cloning and sequencing of the gene encoding a 31-kDa Treponema pallidum subsp. pallidum rare outer membrane porin protein, designated Tromp1 (D. R. Blanco), C. I. Champion, M. M. Exner, H. Erdjument- Bromage, R. E. W. Hancock, P. Tempst, J. N. Miller, and M. A. Lovett, J. Bacteriol. 177:3556-3562, 1995). Here, we report the stable expression of recombinant Tromp1 (rTromp1) in Escherichia coli. rTromp1 expressed without its signal peptide and containing a 22-residue N-terminal fusion resulted in high-level accumulation of a nonexported soluble protein that was purified to homogeneity by fast protein liquid chromatography (FPLC). Specific antiserum generated to the FPLC-purified rTromp1 fusion identified on immunoblots of T. pallidum the native 31-kDa Tromp1 protein and two higher-molecular-mass oligomeric forms of Tromp1 at 55 and 80 kDa. rTromp1 was also expressed with its native signal peptide by using an inducible 17 promoter. Under these conditions, rTromp1 fractionated predominantly with the E. coli soluble and outer membrane fractions, but not with the inner membrane fraction. rTromp1 isolated from the E. coli outer membrane and reconstituted into planar lipid bilayers showed porin activity based on average single-channel conductances of 0.4 and 0.8 nS in 1 M KCl. Whole-mount immunoelectron microscopy using infection-derived immune serum against T. pallidum indicated that rTromp1 was surface exposed when expressed in E. coli. These findings demonstrate that rTromp1 can be targeted to the E. coli outer membrane, where it has both porin activity and surface antigenic exposure.

LanguageEnglish
Pages6685-6692
Number of pages8
JournalJournal of bacteriology
Volume178
Issue number23
DOIs
Publication statusPublished - 1 Jan 1996

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

Cite this

Blanco, David R. ; Champion, Cheryl I. ; Exner, Maurice M. ; Shang, Ellen S. ; Skare, Jonathan T. ; Hancock, Robert ; Miller, James N. ; Lovett, Michael A. / Recombinant Treponema pallidum rare outer membrane protein 1 (Tromp1) expressed in Escherichia coli has porin activity and surface antigenic exposure. In: Journal of bacteriology. 1996 ; Vol. 178, No. 23. pp. 6685-6692.
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abstract = "We recently reported the cloning and sequencing of the gene encoding a 31-kDa Treponema pallidum subsp. pallidum rare outer membrane porin protein, designated Tromp1 (D. R. Blanco), C. I. Champion, M. M. Exner, H. Erdjument- Bromage, R. E. W. Hancock, P. Tempst, J. N. Miller, and M. A. Lovett, J. Bacteriol. 177:3556-3562, 1995). Here, we report the stable expression of recombinant Tromp1 (rTromp1) in Escherichia coli. rTromp1 expressed without its signal peptide and containing a 22-residue N-terminal fusion resulted in high-level accumulation of a nonexported soluble protein that was purified to homogeneity by fast protein liquid chromatography (FPLC). Specific antiserum generated to the FPLC-purified rTromp1 fusion identified on immunoblots of T. pallidum the native 31-kDa Tromp1 protein and two higher-molecular-mass oligomeric forms of Tromp1 at 55 and 80 kDa. rTromp1 was also expressed with its native signal peptide by using an inducible 17 promoter. Under these conditions, rTromp1 fractionated predominantly with the E. coli soluble and outer membrane fractions, but not with the inner membrane fraction. rTromp1 isolated from the E. coli outer membrane and reconstituted into planar lipid bilayers showed porin activity based on average single-channel conductances of 0.4 and 0.8 nS in 1 M KCl. Whole-mount immunoelectron microscopy using infection-derived immune serum against T. pallidum indicated that rTromp1 was surface exposed when expressed in E. coli. These findings demonstrate that rTromp1 can be targeted to the E. coli outer membrane, where it has both porin activity and surface antigenic exposure.",
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Recombinant Treponema pallidum rare outer membrane protein 1 (Tromp1) expressed in Escherichia coli has porin activity and surface antigenic exposure. / Blanco, David R.; Champion, Cheryl I.; Exner, Maurice M.; Shang, Ellen S.; Skare, Jonathan T.; Hancock, Robert; Miller, James N.; Lovett, Michael A.

In: Journal of bacteriology, Vol. 178, No. 23, 01.01.1996, p. 6685-6692.

Research output: Contribution to journalArticle

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T1 - Recombinant Treponema pallidum rare outer membrane protein 1 (Tromp1) expressed in Escherichia coli has porin activity and surface antigenic exposure

AU - Blanco, David R.

AU - Champion, Cheryl I.

AU - Exner, Maurice M.

AU - Shang, Ellen S.

AU - Skare, Jonathan T.

AU - Hancock, Robert

AU - Miller, James N.

AU - Lovett, Michael A.

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